Microextraction is a significant departure from conventional ‘sampling’ techniques. In conventional sampling techniques, a portion of the system under study is removed from its natural environment and the compounds of interest extracted and analyzed in a laboratory environment. In microextraction, compounds of interest are not exhaustively removed from the investigated system, and conditions can be devised where only a small proportion of the total amount of compound, and none of the matrix, are removed. This avoids disturbing the normal balance of chemical components. Because extracted chemicals can be separated chromatographically and quantified by highly sensitive analytical instruments, high accuracy, sensitivity and selectivity are achieved.
With current commercially available solid phase microextraction (SPME) devices, a stationary extraction phase is coated onto a fused silica fibre. The coated portion of the fibre is typically about 1 cm long and coatings have various thicknesses. The fibre can be mounted into a stainless steel support tube and housed in a syringe-like device for ease of use. Extractions are performed by exposing the extraction phase to a sample for a pre-determined time to allow sample components to come into equilibrium with the extraction phase. After extraction, the fibre is removed to an analytical instrument (typically a gas or liquid chromatograph) where extracted components are desorbed and analysed. The amount of a component extracted is proportional to its concentration in the sample (J. Pawliszyn “Method and Device for Solid Phase Microextraction and Desorption”, U.S. Pat. No. 5,691,206.).
To date, commercial SPME devices have been used in some applications of direct analysis of living systems. For example they have been applied for the analysis of airborne pheromones and semiochemicals used in chemical communications by insects (Moneti, G.; Dani, F. R.; Pieraccini, G. T. S. Rapid Commun. Mass Spectrom. 1997, 11, 857-862.), (Frerot, B.; Malosse, C.; Cain, A. H. J. High Resolut. Chromatogr. 1997, 20, 340-342.) and frogs (Smith, B. P.; Zini, C. A.; Pawliszyn, J.; Tyler, M. J.; Hayasaka, Y.; Williams, B.; Caramao, E. B. Chemistry and Ecology 2000, 17, 215-225.) respectively. In these cases, the living animals were non-invasively monitored over time by assessing the chemical concentrations in the air around the animal, providing a convenient means to study complicated dynamic processes without interference.
The current commercial devices do, however, have some limitations for in vivo and in vitro analysis of a biological matrix, such as blood or tissue. Firstly, the most difficult and undesirable problem is the adsorption of proteins and other macromolecules on the surface of SPME fibres. Devices can be made biocompatible by coating them with a biocompatible material. Custom-made coatings based on polypyrrole (PPY) (Lord, H. L.; Grant, R. P.; Walles, M.; Incledon, B.; Fahie, B.; Pawliszyn, J. B., Anal. Chem. 2003, 75(19), 5103-5115) and poly(ethylene glycol) (PEG) (Musteata, F. M.; Musteata, M. L.; Pawliszyn, J., Clin Chem 2006, 52(4), 708-715) have been used for in vivo drug analysis.
Other materials which have been used to reduce the adsorption of proteins and other macromolecules found in a biological matrix include: restricted access materials (RAM), ionic liquids (IL), polydimethylsiloxane (PDMS), polypyrrole, and poly(ethylene glycol). Biocompatible membranes have also been prepared from polyacrylonitrile (Nie, F.-Q.; Xu, Z.-K.; Ming, Y.-Q.; Kou, R.-Q.; Liu, Z.-M.; Wang, S.-Y. Desalination 2004, 160, 43-50. Lavaud, S.; Canivet, E.; Wuillai, A.; Maheut, H.; Randoux, C.; Bonnet, J.-M.; Renaux, J.-L.; Chanard, J. Nephrology, Dialysis, Transplantation 2003, 18, 2097-2104. Yang, M. C.; Lin, W. C. Journal of Polymer Research 2002, 9, 201-206), polyurethane, chitosan, and cellulose.
As noted above, in analysis of chemicals in a biological matrix, such as blood or tissue, the most difficult and undesirable problem is the adsorption of proteins and other macromolecules on the surface of SPME fibres. In contrast, in the analysis of chemicals in vegetable, fruit or food processing, such as fruit juices, the most difficult and undesirable problem is the adsorption of carbohydrates on the surface of SPME fibres. Adsorbed carbohydrates may be transformed into carbon deposits when heated in analytical instruments, such when used for gas chromatography.
Conventional methods of sample preparation used in analysis of pesticides residues in vegetable, fruit and food processing may include liquid-liquid extractions, microwave assisted extraction, supercritical fluid extraction, on-line microextraction, or solid-phase extraction. These methods can be time-consuming, tedious and hazardous to operators' health, for example due to the organic solvents involved.
Another method used in analysis of pesticides residues in vegetable, fruit and food processing includes an extraction procedure based on liquid-liquid partitioning with acetonitrile followed by a cleanup step with dispersive-SPE (solid phase extraction), was described by Anastassiades et al. (Journal of AOAC International. 2003, Vol. 86, 2, pp. 412-431). This method has been applied in multi-residue analysis of pesticides in fruits and vegetables. However, the method is a multistep method and cannot be automated. The combination of sample preparation and instrument introduction steps is not accomplished by this method. Furthermore, this sample preparation technique uses multistep, labor-intensive procedure and requires the use of organic solvents.
It is desirable to develop an automated methodology to analyze pesticides in food matrices, for example using a high sample throughput with the entire analysis being completely automated. Solid-phase microextraction (SPME), which integrates sampling, extraction, concentration and sample introduction into a single step may be used to facilitate rapid sample preparation and integrate sampling, extraction, concentration and sample introduction to an analytical instrument into one solvent-free step. However, the facilitation of high-quality analytical methods in combination with SPME requires optimization of the parameters that affect the extraction efficiency; namely, extraction phase chemistry, extraction mode, agitation method, sample modification (pH, ionic strength, organic solvent content), sample temperature, extraction time and desorption conditions (Nature Protocols. 2010, Vol. 5, 1, pp. 122-139).
SPME has been used in the extraction of pesticides residues in vegetal foodstuff. However, food applications usually use headspace-SPME (HS-SPME), in which the extraction phase is placed in the headspace above the sample, rather than immersed into the sample. One limitation of this approach is that the rates of extraction are low for poorly volatile or polar analytes.
Another SPME method which has been used is direct immersion SPME (DI-SPME). Due to the complex nature of food matrices, DI-SPME can be difficult and is typically a poor choice for food analysis, especially when using solid sorbents (L. S De Jager, G. A. Perfetti, G. W. Diachenko. Analysis of tetramethylene disulfotetramine in foods using solid-phase microextraction-gas chromatography-mass spectrometry. Journal of Chromatography A. 2008, Vol. 1192, pp. 36-40). DI-SPME can be difficult because pretreatment of the sample may be necessary to protect the coating and avoid the fouling of the extraction phase by irreversible adsorption of macromolecules from the complex matrix at the interface. Such fouling could lead to a substantial decrease in the fibre lifetime, making it unusable for more than a few samples, and could also change the coating extraction properties (Journal of Chromatography A. 2007, Vol. 1153, pp. 36-53). The additional pretreatment or clean-up prior to SPME extraction may include, for example, centrifugation, dilution or pre-extraction in organic solvent.
It is desirable to provide a SPME fibre to be used in SPME, for example in DI-SPME or HS-SPME, where the SPME fibre is able to extract small molecules from a vegetable, fruit or food matrix, and where the SPME fibre has a protective coating which reduces adsorption of carbohydrates on the surface of the coated SPME fibres in comparison to a non-protectively coated SPME fibre. Additionally, it is desirable to provide a coating which is stable to chromatographic techniques used to analyze the extracted small molecules. Additionally, it is desirable to provide a process for coating an SPME fibre with said such a protective coating.